Methods for amplifying nucleic acids provide useful tools for the detection of human pathogens, detection of human genetic polymorphisms, detection of RNA and DNA sequences, for molecular cloning, sequencing of nucleic acids, and the like. In particular, the polymerase chain reaction (PCR) has become an important tool in the cloning of DNA sequences, forensics, paternity testing, pathogen identification, disease diagnosis, and other useful methods where the amplification of a nucleic acid sequence is desired. See e.g., PCR Technology: Principles and Applications for DNA Amplification (Erlich, ed., 1992); PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990).
PCR permits the copying, and resulting amplification, of a target nucleic acid. Briefly, a target nucleic acid, e.g. DNA, is combined with a sense and antisense primers, dNTPs, DNA polymerase and other reaction components. See Innis et al. The sense primer can anneal to the antisense strand of a DNA sequence of interest. The antisense primer can anneal to the sense strand of the DNA sequence, downstream of the location where the sense primer anneals to the DNA target. In the first round of amplification, the DNA polymerase extends the antisense and sense primers that are annealed to the target nucleic acid. The first strands are synthesized as long strands of indiscriminate length. In the second round of amplification, the antisense and sense primers anneal to the parent target nucleic acid and to the complementary sequences on the long strands. The DNA polymerase then extends the annealed primers to form strands of discrete length that are complementary to each other. The subsequent rounds serve to predominantly amplify the DNA molecules of the discrete length.
A variety of factors can lead to non-functional PCR or other amplification reactions. One drawback of PCR is that artifacts can be generated from mis-priming and primer dimerization. Those artifacts can be exacerbated in traditional multiplex PCR. Multiple sets of primers increase the possibility of primer complementarity at the 3xe2x80x2-ends, leading to primer-dimer formation. These artifacts deplete the reaction of dNTPs and primers and outcompete the multiplex templates for DNA polymerase. Such artifacts can be reduced by careful primer design and the use of xe2x80x9chot startxe2x80x9d PCR. See Chou, Q. et al. (1992) Nucleic Acids Research, 20: 1717-1723. It is increasingly difficult, however, to eliminate all interactions which promote the mis-priming and primer dimerization in a multiplex amplification as the reaction may contain many primers at high concentration.
Additionally, multiplex PCR has been observed to suppress the amplification of one template in preference for another template. A number of factors are involved in this suppression. For example, when a multiplex PCR reaction involves different priming events for different target sequences, the relative efficiency of these events may vary for different targets. This can be due to the differences in thermodynamic structure, stability, and hybridization kinetics among the various primers used.
Simple user error, of course, can also result in a nonfunctional amplification reaction. For instance, the absence of nucleotides or enzyme due to negligence or degradation will lead to a nonfunctional reaction. Similarly, where probes are used to monitor a particular reaction, a nonfunctional probe will lead to a false negative reaction. This can occur, for instance, when there is an absence of probe or the probe does not bind to its hybridization site efficiently. Use of probes, particularly fluorescent probes, are commonly used for monitoring the accumulation of reaction products in real time, i.e. while that amplification reaction is progressing.
Several schemes for controlling for failure of an amplification reaction have been described. See, e.g., Edwards, M., et al. PCR PRIMER, A LABORATORY MANUAL (Dieffenbach, C., et al., eds. 1995) pages 157-171. For example, it is common to run positive and negative control reactions in separate reaction tubes. Simple positive controls include a known amount of template, while negative controls do not have any template in the reaction. These controls are run under the same conditions as a test sample and provide the tester with information about the quality of the enzymes and nucleotides, etc., as well as whether the test solutions are contaminated.
More recently, internal controls for PCR have been developed. Internal controls are advantageous because they are run in the exact same reaction mixture as the test sample and therefore there is no question about the activity of the reagents in the test sample itself. Moreover, internal controls are more efficient by allowing for the use of fewer reactions and less reaction solution and reagents.
Internal controls typically involve multiple reactions performed in the same reaction tube (e.g., multiplex PCR). In such reactions, the presence of at least one amplification product indicates that some variables, such as the enzyme and nucleotides, were functional during the reaction. See, e.g. Levinson, G. et al. Human Reprod. 7(9):1304-1313 (1992).
In addition, internal controls to verify the presence of the target template have also been described. For example, in multiplex assays where closely related templates such as pathogen strains are distinguished by amplifying differing sequences, primers for a sequence common to all templates provides a positive control for amplification. See, e.g., Kaltenboeck, B., et al. J. Clin. Microbiol. 30(5):1098-1104 (1992); Way, J., et al. App. Environ. Microbiol. 59(5):1473-1479 (1993); Wilton, S. et al. PCR Methods Appl. 1:269-273 (1992). Rosenstraus et al. (J. Clin. Microbiol. 36(1):191-197 (1998)) have described an internal control containing primer binding regions identical to those of the target sequence and that contain a unique probe-binding region that differentiates the control from the amplified target sequence.
As discussed above, it is often desirable to quantify PCR products using various fluorescent probes. Examples of useful fluorescent probes include, e.g., fluorescence resonance energy transfer (FRET), molecular beacon, and TaqMan(copyright) probes. Currently, however, there is no internal control method that validates the activity of a target specific probe in the same reaction mixture as the test sample. Therefore, to have a fully validated amplification reaction, a positive control must be run in a separate reaction tube to insure that the target specific probe is functioning properly.
Accordingly, there is a need for internal control compositions and methods useful for measuring these and other amplification variables. The present invention meets this need and provides useful methods and compositions for performing a totally internally controlled amplification reaction.
The present invention provides methods of performing an amplification reaction. The steps of the reaction comprise:
(a) combining in an aqueous solution,
(i) a target probe, a first control probe and a second control probe;
(ii) a first 5xe2x80x2 primer, a first 3xe2x80x2 primer and a target template, the target template comprising a hybridization site for the first 5xe2x80x2 primer, the first 3xe2x80x2 primer and the target probe;
(iii) a first control template, the first control template comprising a hybridization site for the first 5xe2x80x2 primer, the first 3xe2x80x2 primer and the first control probe; and
(iv) a second 5xe2x80x2 primer, a second 3xe2x80x2 primer and a second control template, the second control template comprising a hybridization site for the second 5xe2x80x2 primer, the second 3xe2x80x2 primer, the target probe and a second control probe;
(b) performing an amplification reaction; and
(c) quantifying binding of the target probe, first control probe and second control probe.
In some embodiments, the quantifying step is performed during the amplification reaction. In some embodiments, the quantifying step is performed after the amplification reaction.
In some embodiments, the target probe, first control probe and second control probe comprise a fluorophore. For example, the target probe, first control probe and second control probe can each comprise a fluorophore which fluoresces at a different wavelength of light. In some embodiments, the target probe, first control probe and second control probe further comprise a quenching reagent. In some embodiments, the target probe, first control probe and the second control probe are enzymatically cleaved during the amplification reaction. For instance, the quenching reagent and the fluorophore can be separated when the probes hybridize to their hybridization sites.
In some embodiments, the amplification reaction is a thermocyclic amplification reaction. For example, the thermocyclic amplification reaction can be a polymerase chain reaction (PCR). In some embodiments, the amplification reaction is an isothermal reaction. For example, the isothermal reaction can be a transcription-mediated amplification (TMA).
The present invention also provides kits comprising:
(a) a target probe, a first control probe and a second control probe;
(b) a first 5xe2x80x2 primer and a first 3xe2x80x2 primer;
(c) a first control template, the first control template comprising a hybridization site for the first 5xe2x80x2 primer, the first 3xe2x80x2 primer and the first control probe; and
(d) a second 5xe2x80x2 primer, a second 3xe2x80x2 primer and a second control template, the second control template comprising a hybridization site for the second 5xe2x80x2 primer, the second 3xe2x80x2 primer, the target probe and the second control probe.
In some embodiments, the kit further comprises nucleotides and a DNA polymerase. In some embodiments, the target probe, first control probe and second control probe comprise a fluorophore. For example, the target probe, first control probe and second control probe can further comprise a quenching reagent.
The present invention also provides a solution comprising:
(a) a target probe, a first control probe and a second control probe;
(b) a first 5xe2x80x2 primer and a first 3xe2x80x2 primer;
(c) a first control template, the first control template comprising a hybridization site for the first 5xe2x80x2 primer, the first 3xe2x80x2 primer and the first control probe; and
(d) a second 5xe2x80x2 primer, a second 3xe2x80x2 primer and a second control template, the second control template comprising a hybridization site for the second 5xe2x80x2 primer, the second 3xe2x80x2 primer, the target probe and the second control probe.
In some embodiments, the solution further comprises nucleotides and a DNA polymerase. Moreover, the target probe, first control probe and second control probe can comprise a fluorophore. In addition, the target probe, first control probe and second control probe further comprise a quenching reagent.
An xe2x80x9camplification reactionxe2x80x9d refers to any chemical, including enzymatic, reaction that results in increased copies of a template nucleic acid sequence. Amplification reactions include polymerase chain reaction (PCR) and ligase chain reaction (LCR) (see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)), strand displacement amplification (SDA) (Walker, et al. Nucleic Acids Res. 20(7):1691-6 (1992); Walker PCR Methods Appl 3(1):1-6 (1993)), transcription-mediated amplification (Phyffer, et al., J. Clin. Microbiol. 34:834-841 (1996); Yuorinen, et al., J. Clin. Microbiol. 33:1856-1859 (1995)), nucleic acid sequence-based amplification (NASBA) (Compton, Nature 350(6313):91-2 (1991), rolling circle amplification (RCA) (Lisby, Mol. Biotechnol. 12(1):75-99 (1999)); Hatch et al., Genet. Anal. 15(2):35-40 (1999)) and branched DNA signal amplification (bDNA) (see, e.g., Iqbal et al., Mol. Cell Probes 13(4):315-320 (1999)).
A xe2x80x9cthermocyclic amplification reactionxe2x80x9d refers to the amplification of DNA fragments by using primer oligonucleotides which, with the aid of a thermostable enzyme, synthesizes or ligates copies a template nucleic acid sequence. Thermocyclic reactions such as the polymerase chain reaction (PCR) and the ligase chain reaction (LCR) are well known.
A xe2x80x9ctargetxe2x80x9d or xe2x80x9ctarget nucleic acidxe2x80x9d refers to a single or double stranded polynucleotide sequence sought to be amplified in a thermocyclic amplification reaction.
A xe2x80x9cprobexe2x80x9d refers to a polynucleotide sequence capable of hybridization to a polynucleotide sequence of interest and allows for the detecting of the polynucleotide sequence of choice. For example, xe2x80x9cprobesxe2x80x9d can comprise polynucleotides linked to fluorescent or radioactive reagents, thereby allowing for the detection of these reagents.
A xe2x80x9ctemplatexe2x80x9d refers to a double stranded polynucleotide sequence that comprises the polynucleotide to be amplified, flanked by primer hybridization sites. Thus, a xe2x80x9ctarget templatexe2x80x9d comprises the target polynucleotide sequence flanked by hybridization sites for a 5xe2x80x2 target primer and a 3xe2x80x2 target primer. A xe2x80x9ccontrol templatexe2x80x9d refers to a polynucleotide sequence that is flanked by primer hybridization sequences.
A xe2x80x9c5xe2x80x2 target primerxe2x80x9d refers to a polynucleotide sequence, typically less than 100 nucleotides in length, which hybridizes to a sequence on the target template that is upstream from the target polynucleotide. A xe2x80x9c3xe2x80x2 target primerxe2x80x9d refers to a polynucleotide sequence, typically less than 100 nucleotides in length, whose complement hybridizes to a sequence on the target template downstream from the target polynucleotide.
A xe2x80x9chybridization sitexe2x80x9d refers to a polynucleotide sequence that is the complement for a primer or probe polynucleotide sequence and that can be bound by a specific primer or probe.
xe2x80x9cQuantifying bindingxe2x80x9d refers to measuring the absolute or relative binding of probe molecules to regions of nucleic acid sequence to which they hybridize. For instance, binding of fluorescent probes using FRET technology can be measured by quantifying the amount of light emitted from a sample at a particular wavelength. Alternatively, binding of probes using standard blotting and hybridization techniques (e.g., Southern blotting) can be quantified with a variety of computer-based scanning devices.
xe2x80x9cSubstantially equal concentrationsxe2x80x9d means that the concentration of one solute is from 95% to 105% of the concentration of at least one other solute in a solution.
The term xe2x80x9cfluorophorexe2x80x9d refers to chemical compounds which, when excited by exposure to particular wavelengths of light, emit light (i.e. fluoresce) at a different wavelength.
When the excited-state energy of the fluorophore is transferred to a non-fluorophore acceptor, the fluorescence of the fluorophore is quenched without subsequent emission of fluorescence by the acceptor. In this case, the acceptor functions as a xe2x80x9cquenching agentxe2x80x9d.
The phrase xe2x80x9cnucleic acidxe2x80x9d refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
The following abbreviations are provided for the reader""s convenience:
Target=T
Internal control 1 (or first control template)=IC1
Internal control 2 (or second control template)=IC2
Forward (or first 5xe2x80x2) primer for target and IC1=P1
Reverse (or first 3xe2x80x2) primer for target and IC1=P2
Forward (or second 5xe2x80x2) primer for IC2=P3
Reverse (or second 3xe2x80x2 ) primer for IC2=P4
Hybridization probe for the target and IC2 (xe2x80x9ctarget probexe2x80x9d)=HP1
Hybridization probe for IC1 (xe2x80x9cfirst control probexe2x80x9d)=HP2
Hybridization probe for IC2 (xe2x80x9csecond control probexe2x80x9d)=HP3